Abstract
Overexpression and activation of the c-Src protein have been linked to the development of a wide variety of cancers. The molecular mechanism(s) of c-Src overexpression in cancer cells is not clear. We report here an internal ribosome entry site (IRES) in the c-Src mRNA that is constituted by both 5'-noncoding and -coding regions. The inhibition of cap-dependent translation by m(7)GDP in the cell-free translation system or induction of endoplasmic reticulum stress in hepatoma-derived cells resulted in stimulation of the c-Src IRES activities. Sucrose density gradient analyses revealed formation of a stable binary complex between the c-Src IRES and purified HeLa 40 S ribosomal subunit in the absence of initiation factors. We further demonstrate eIF2-independent assembly of 80 S initiation complex on the c-Src IRES. These features of the c-Src IRES appear to be reminiscent of that of hepatitis C virus-like IRESs and translation initiation in prokaryotes. Transfection studies and genetic analysis revealed that the c-Src IRES permitted initiation at the authentic AUG351, which is also used for conventional translation initiation of the c-Src mRNA. Our studies unveiled a novel regulatory mechanism of c-Src synthesis mediated by an IRES element, which exhibits enhanced activity during cellular stress and is likely to cause c-Src overexpression during oncogenesis and metastasis.
Highlights
The internal ribosome entry site (IRES) elements have been detected in a number of eukaryotic mRNAs that encode proteins involved in signal transduction pathways, gene expression and development, differentiation, apoptosis, and cell cycle or stress response [1, 2, 7]
To specify the position of the free probes during centrifugation. c-Src mRNA Motif Supports Cap-independent Translation of Reporter RNAs—firefly luciferase (FLuc)-based reporter mRNAs were engineered to test if the c-Src 5Ј-noncoding region (5ЈNCR) supports cap-independent translation
Unlike known cellular IRESs, the c-Src IRES demonstrated here exhibits many unique attributes that are analogous to the characteristics of HCV-like IRESs
Summary
Plasmid Constructs—Total RNA was isolated from the hepatoma-derived Huh cell line using Qiagen RNeasy kit. The c-Src PCR-amplified DNA described above was digested with HindIII and NcoI and cloned at the same site in the vector backbone In both cases, the Kozak sequence context was maintained at the translation site. RNA Stability Assay—Equal amounts of 32P-labeled wild type or mutant reporter RNAs were incubated in standard HeLa translation reactions, and total RNAs were extracted from each sample using the RNeasy kit (Qiagen). IRES or a nonspecific RNA probe derived from 5ЈPV(⌬286 – AUG, which conforms with Py tracts found in many viral IRESs. 605) as scrambled IRES was mixed with purified HeLa 40 S These characteristics and the Y-shaped architectural features subunit in buffer K containing 20 units of RNasin in a final are considered as important elements of many viral and cellular volume of 40 l and incubated for 15 min at 30 °C. To specify the position of the free probes during centrifugation. c-Src mRNA Motif Supports Cap-independent Translation of
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